Tendon Regeneration: Understanding Tissue Physiology and Development to Engineer Functional Substitutes

Tendon Regeneration

Understanding Tissue Physiology and Development to Engineer Functional Substitutes

Editors

Manuela E. Gomes

Rui L. Reis

Márcia T. Rodrigues

Table of Contents

Cover image

Title page

Copyright

Contributors

Preface

Section 1. Biology and Physiology of Tendons

Chapter 1. Tendon Physiology and Mechanical Behavior: Structure–Function Relationships

1. Tendon Structure and Composition

2. Tendon Mechanics

3. Multiscale Mechanics and Structure–Function Characterization

4. Mechanical and Compositional Variations in Tendons with Different Functions

List of Abbreviations

Glossary

Chapter 2. Tendon Resident Cells—Functions and Features in Section I—Developmental Biology and Physiology of Tendons

1. Introduction

2. Tendon Cells—Origin and Specification

3. Tendon Cells—ECM Synthesis, Assembly, and Tissue Maturation

4. Cell–ECM Interactions

5. Mechanoregulation of Tendon Cells

6. Conclusion

List of Abbreviations

Glossary

Chapter 3. Mechanobiology of Embryonic and Adult Tendons

1. Introduction

2. Embryonic Tendon

3. Postnatal Tendon

4. Mechanical Cues Experienced by Embryonic, Postnatal, and Adult Tendons

5. Studies in the Embryo Suggest Mechanical Factors Influence Embryonic Tendon Development

6. In Vitro Studies Suggest Mechanical Factors Influence Embryonic Tendon Development

7. Exercise Studies Examine the Influence of Mechanics in Adult Tendon

8. In Vitro Studies Suggest Mechanical Factors Influence Adult Tendon Homeostasis

9. Potential Mechanisms of Tendon Cell Mechanotransduction

10. Conclusions

List of Abbreviations

Section 2. Pathologies and Repair of Tendons

Chapter 4. Tendinopathy I: Understanding Epidemiology, Pathology, Healing, and Treatment

1. Introduction

2. Anatomical Diagnosis

3. Pathology

4. Epidemiology

5. Pathophysiology

6. Healing and Repair

7. Nonsurgical Treatment

8. Surgical Treatment

9. Conclusion

List of Abbreviations

Glossary

Chapter 5. Tendinopathy II: Etiology, Pathology, and Healing of Tendon Injury and Disease

1. Epidemiology

2. Definitions

3. Tendinopathy Etiology

4. Pathology

5. Summary and Conclusions

List of Abbreviations

Glossary

Section 3. Tendon Regenerative Medicine Approaches

Chapter 6. Cell-Based Approaches for Tendon Regeneration

1. Introduction

2. Tendon Endogenous Regeneration

3. Isolation Procedures of Tendon Resident Cells

4. Alternative Stem Cells Sources for Cell-Based Tendon Tissue Engineering

5. Moving Cell Therapies into the Clinics

6. Conclusion

List of Abbreviations

Glossary

Chapter 7. The Role of Growth Factors in Tendon Stimulation

1. Introduction

2. Growth Factors

3. Platelet-Rich Plasma

4. Conclusions

List of Abbreviations

Section 4. Scaffolds-Based Approaches

Chapter 8. Engineering Anisotropic 2D and 3D Structures for Tendon Repair and Regeneration

1. Introduction

2. Anisotropic Sponges

3. Anisotropic Self-Assembled Fibers

4. Anisotropic Electrospun Fibers

5. Anisotropic Imprinted Substrates

6. Conclusive Remarks

List of Abbreviations

Glossary

Chapter 9. Biologic- and Synthetic-Based Scaffolds for Tendon Regeneration

1. Tendon Injuries

2. Tendon Repair and Tissue Engineering Strategies

3. Criteria and Requirements for Tendon Tissue Engineering Scaffolds

4. Types for Tendon Tissue Engineering Scaffolds

5. Scaffold Architecture and Design

6. Functional and Bioactive Scaffolds

7. Conclusions

List of Abbreviations

Section 5. Tendon Tissue Engineering

Chapter 10. Fabrication of Hierarchical and Biomimetic Fibrous Structures to Support the Regeneration of Tendon Tissues

1. Introduction

2. Spinning Techniques for Tendon TE Scaffolding

3. Rapid Prototyping Technique

4. Electrochemically Aligned Collagen

5. Microengineered Hydrogels

6. Assembly of Fibrous Biomaterials into Higher Hierarchical Structures

7. Concluding Remarks and Future Perspectives

List of Abbreviations

Glossary

Chapter 11. Multifactorial Tendon Tissue Engineering Strategies

1. Introduction

2. Cells, Scaffolds, and Mechanical Stimulation

3. Approaches for Different Anatomical Regions

4. Design of Custom Bioreactors

5. Concluding Remarks

List of Abbreviations

Glossary

Chapter 12. Tendon Tissue Engineering: Combined Tissue Engineering Approach for the Regeneration of Tendons

1. Introduction

2. Functional Tendon Tissue Engineering

3. Tendon Tissue Engineering Bioreactors and Construct Stimulation

4. Conclusions

List of Abbreviations

Glossary

Chapter 13. Biomaterial Scaffolds for Tendon Tissue Engineering

1. Motivation: Tendon Injury and Repair Mechanism

2. Introduction to Tissue Engineering

3. Cell–Biomaterial Interactions

4. Clinical Translation and Adaptation to Complex Musculoskeletal Injury Models

5. Conclusions

List of Abbreviations

Glossary

Chapter 14. Engineered Tendon Repair and Regeneration

1. Introduction

2. Cell Sources in Tendon Engineering and Regeneration

3. Scaffold Materials for Tendon Engineering

4. In Vivo, In Vitro, and Ex Vivo Tendon Engineering

5. Conclusion

List of Abbreviations

Glossary

Chapter 15. Scaffold Design for Integrative Tendon–Bone Repair

1. Introduction

2. Rotator Cuff Tendon Augmentation Grafts

3. Integrative Rotator Cuff Tendon–Bone Repair

4. Summary and Future Directions

List of Abbreviations

Index

Copyright

Academic Press is an imprint of Elsevier

125 London Wall, London EC2Y 5AS, UK

525 B Street, Suite 1800, San Diego, CA 92101-4495, USA

225 Wyman Street, Waltham, MA 02451, USA

The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK

Copyright © 2015 Elsevier Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

ISBN: 978-0-12-801590-2

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library

Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress

For information on all Academic Press publications visit our website at http://store.elsevier.com/

Publisher: Mica Haley

Acquisition Editor: Mica Haley

Editorial Project Manager: Lisa Eppich

Production Project Manager: Julia Haynes

Designer: Inês Cruz

Typeset by TNQ Books and Journals

www.tnq.co.in

Printed and bound in the United States of America

Contributors

Paul W. Ackermann,     Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden

Giuseppe Banfi

Dipartimento di Scienze Biomediche per la Salute, Università degli Studi di Milano, Milan, Italy

IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy

Manus Biggs

Network of Excellence for Functional Biomaterials (NFB), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Centre for Research in Medical Devices (CURAM), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Helen L. Birch,     Institute of Orthopaedics and Musculoskeletal Science, University College London, Stanmore, UK

Paolo Cabitza

Dipartimento di Scienze Biomediche per la Salute, Università degli Studi di Milano, Milan, Italy

IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy

Yilin Cao,     Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Tissue Engineering Key Laboratory, National Tissue Engineering Center of China, Shanghai, P.R. China

Peter D. Clegg,     Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst Campus, Neston, UK

Raquel Costa-Almeida

3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães, Portugal

Riccardo D’Ambrosi,     IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy

Rui M.A. Domingues

3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães, Portugal

Alicia J. El Haj,     Institute of Science and Technology in Medicine, Keele University Medical School, Guy Hilton Research Centre, University Hospital North Midlands, North Staffs, UK

Brandon Engebretson,     School of Chemical, Biological, and Materials Engineering, University of Oklahoma, Norman, OK, USA

Andrew English

Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Network of Excellence for Functional Biomaterials (NFB), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Centre for Research in Medical Devices (CURAM), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Pavel Gershovich

3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães, Portugal

Manuela E. Gomes

3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães, Portugal

Ana I. Gonçalves

3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães, Portugal

Brendan Harley

Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Laura A. Hockaday,     Department of Biomedical Engineering, Tufts University, Medford, MA, USA

Rebecca Hortensius,     Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Faith W. Karanja,     Cell, Molecular and Developmental Biology Program, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA

Catherine K. Kuo

Department of Biomedical Engineering, Tufts University, Medford, MA, USA

Cell, Molecular and Developmental Biology Program, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA, USA

Thomas D. Kwan,     Institute of Science and Technology in Medicine, Keele University Medical School, Guy Hilton Research Centre, University Hospital North Midlands, North Staffs, UK

William N. Levine,     Department of Orthopaedic Surgery, Columbia University, New York Presbyterian Hospital, New York, NY, USA

Wei Liu,     Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Tissue Engineering Key Laboratory, National Tissue Engineering Center of China, Shanghai, P.R. China

Alex Lomas

Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Network of Excellence for Functional Biomaterials (NFB), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Centre for Research in Medical Devices (CURAM), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Helen H. Lu,     Biomaterials and Interface Tissue Engineering Laboratory, Department of Biomedical Engineering, Columbia University, New York, NY, USA

Alessandra Menon,     IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy

Tyler R. Morris,     McKay Orthopaedic Research Laboratory, University of Pennsylvania, Philadelphia, PA, USA

Laura Mozdzen,     Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Zachary Mussett,     School of Biomedical Engineering, University of Oklahoma, Norman, OK, USA

Abhay Pandit

Network of Excellence for Functional Biomaterials (NFB), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Centre for Research in Medical Devices (CURAM), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Vincenza Ragone,     IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy

Filippo Randelli,     IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy

Pietro Randelli,     IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy

Rui L. Reis

3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães, Portugal

Corinne N. Riggin,     McKay Orthopaedic Research Laboratory, University of Pennsylvania, Philadelphia, PA, USA

Márcia T. Rodrigues

3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães, Portugal

Benjamin B. Rothrauff,     Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Mitchell D. Saeger,     Department of Chemical and Biological Engineering, Tufts University, Medford, MA, USA

Sambit Sahoo,     Department of Biomedical Engineering, Cleveland Clinic, Cleveland, OH, USA

Hazel R.C. Screen,     Institute of Bioengineering, School of Engineering & Materials Science, Queen Mary University of London, London, UK

Vassilios Sikavitsas

School of Chemical, Biological, and Materials Engineering, University of Oklahoma, Norman, OK, USA

School of Biomedical Engineering, University of Oklahoma, Norman, OK, USA

Aaron Simmons,     School of Chemical, Biological, and Materials Engineering, University of Oklahoma, Norman, OK, USA

Louis J. Soslowsky,     McKay Orthopaedic Research Laboratory, University of Pennsylvania, Philadelphia, PA, USA

Chavaunne T. Thorpe,     Institute of Bioengineering, School of Engineering & Materials Science, Queen Mary University of London, London, UK

Rocky S. Tuan,     Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Bin Wang,     Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Tissue Engineering Key Laboratory, National Tissue Engineering Center of China, Shanghai, P.R. China

Cortes Williams,     School of Biomedical Engineering, University of Oklahoma, Norman, OK, USA

Guang Yang,     Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Dimitrios I. Zeugolis

Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Network of Excellence for Functional Biomaterials (NFB), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Centre for Research in Medical Devices (CURAM), Biosciences Research Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

Xinzhi Zhang,     Biomaterials and Interface Tissue Engineering Laboratory, Department of Biomedical Engineering, Columbia University, New York, NY, USA

Preface

In a world of scientific and technological advances, the ability to rebuild or recover tissue function at a clinically significant scale would potentially revolutionize therapeutics in biomedicine applications considering a wide spectrum of tissues prone to injury, disease, and degeneration. Tissue engineering and regenerative medicine are recent scientific fields proposing alternative strategies to solve problems and limitations in clinics that are not functionally overcome by current therapies and procedures to achieve the regeneration of damaged tissues.

Promising tools on tissue engineering and regenerative medicine approaches in general and tendon-related strategies in particular are moving forward bringing new insights on the complex regenerative versus repair mechanisms involved.

In recent years, research has focused more attention to tendon tissues, unveiling aspects of tendon’s intrinsic morphology, architecture, and functionality. The pivotal role of tendons in joint mechanics and movement implies well-established natural mechanisms of action under permanent and fine-tuned adjustments to balance the forces and loadings in order to adapt to changes in the environment. Although walking, running, or standing may be simple and easily achieved mechanisms in daily activities, the complex dynamics involved challenges researchers to combine creativity and knowledge aiming at restoring tissue morphology, architecture, and ultimately tissue functionality.

Since tendons are connective tissues being mainly composed by an extracellular matrix (ECM), a supportive structure to sustain and transmit the loadings and strains of tendons, ECM analogs, or substitutes may be an interesting starting point to investigate in a regenerative strategy. Although many scaffolds have been designed using different biomaterials and fabrication methodologies, there is limited success in current scaffold designs as novel approaches imply that biomaterial scaffolds should provide more than temporary architectural support to meet native tendon requirements. It is widely accepted though that it is crucial to learn from native tendons, understanding the biomechanical cues and architectural phenomena so that the structural composition and organization can be replicated and to assist the design of smart and responsive biomaterials with multifunctional parameters with a new level of sophistication in order to provide the best cellular recognition with improved mechanical properties.

The fact that tendon architecture adapts to balance the changes in mechanical stresses and that stress forces are also dependent on the functional role, and consequently on the anatomical site, customization of strategies may be required to fulfill tendon specific requirements and restoration of local functionality.

Moreover, some lesions are more prone to occur in different areas within the tendon but also at the tendon interfaces, namely tendon–bone junction and muscle–tendon enthesis. Thus, gradient scaffolds combining both aspects of the interface tissue may be also useful to treat these lesions.

Ideally, custom-made scaffold would be the preferred choice. A scaffold adjusted to the defect dimensions, to the biomechanical properties, to the anatomical location, and to tissue skeletal maturity would fit all the criteria for a successful scaffold as a temporary template for promoting tendon regeneration.

Ultimately, tendon regeneration involves the complete restoration of morphology, biochemically and biomechanical properties of the tissue which are critically fine tuned to achieve tissue function that is often jeopardized through spontaneous healing and frequently results in the formation of scar tissue. In spite of the growing understanding on the roles of the biological entities, resident or stem cells, on the actuation of bioactive molecules such as growth factors, or on the establishment of tenogenic markers, the temporal and sequential process that defines the biological cascade responsible to modulate cell behavior and guidance toward a successful mechanism of regeneration has not been discovered, and requires additional considerations for the management of tendon pathologies.

Despite the scientific effort in developing and validation of new strategies using the traditional pillars of tissue engineering, alone or in combination, few bioengineered products have successfully reached the market with a slow translation into clinical practice. Up to date, and to editors’ knowledge, no tendon tissue engineered product has been commercialized, with the exception of biological scaffolds, often obtained from mammalian-derived tissues, and synthetic scaffolds commonly used in graft augmentation devices.

The major goal of this book was to update and gather all the information from recent years in the field of tendon tissue regeneration so as to provide a state-of-the-art scientific document covering fundamental aspects of the tendon tissue that must be considered when designing regenerative strategies. Hot topics on recent findings from a developmental biology perspective to current pathologies and treatments have been identified in this volume and could act as a holistic platform for guidance into innovative strategies aiming at tendon regenerative medicine.

With this book, the editors of Tendon Regeneration intend to leave a door open to the continuity of innovative strategies and challenges while sharing ideas from a biologic to a clinical point of view to be developed, designed, updated, or rethought in forthcoming studies under the tendon regeneration thematic, combining perspectives reunited in this publication and beyond.

Manuela E. Gomes

Rui L. Reis

Márcia T. Rodrigues

Section 1

Biology and Physiology of Tendons

Outline

Chapter 1. Tendon Physiology and Mechanical Behavior: Structure–Function Relationships

Chapter 2. Tendon Resident Cells—Functions and Features in Section I—Developmental Biology and Physiology of Tendons

Chapter 3. Mechanobiology of Embryonic and Adult Tendons

Chapter 1

Tendon Physiology and Mechanical Behavior

Structure–Function Relationships

Chavaunne T. Thorpe¹, Helen L. Birch², Peter D. Clegg³,  and Hazel R.C. Screen¹     ¹Institute of Bioengineering, School of Engineering & Materials Science, Queen Mary University of London, London, UK     ²Institute of Orthopaedics and Musculoskeletal Science, University College London, Stanmore, UK     ³Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst Campus, Neston, UK

Abstract

Tendons are composed of highly aligned collagen, arranged along the long axis for tensile strength. The collagen is arranged in a series of hierarchical levels, surrounded at each level with a predominantly noncollagenous matrix of varying composition. This composite arrangement of fibers in matrix results in a tough tissue, resistant to injury. It also enables subtle variations in composition and organization between tendons with functionally distinct roles, optimizing their mechanics. Tendons can broadly be categorized as positional or energy storing in function. Positional tendons are relatively stiff, to efficiently transfer forces from muscle and position limbs. By contrast, energy-storing tendons are less stiff and more elastic, stretching and recoiling with each stride to store and return energy, reducing the energetic cost of locomotion. Multiscale mechanical, compositional, and organizational characterization of tendon is providing insight into structure–function optimization.

Keywords

Collagen; Composite mechanics; Composition; Energy storing; Extracellular matrix; Mechanical properties; Micromechanics; Nanomechanics; Proteoglycans

Contents

1. Tendon Structure and Composition 4

1.1 Collagens 6

1.2 Proteoglycans 6

1.3 Glycoproteins and Other Molecules 8

1.4 Cells 8

1.5 Bone Insertion 9

1.6 Myotendinous Junction 10

2. Tendon Mechanics 10

2.1 In Vitro Mechanical Testing 11

2.2 In Vivo Mechanical Testing 13

2.3 Viscoelasticity 15

3. Multiscale Mechanics and Structure–Function Characterization 17

3.1 Macroscale Mechanics 18

3.2 Microscale Mechanics 19

3.3 Nanoscale Mechanics 21

3.4 Multiscale Structure–Function Mechanistic Studies 22

3.5 Enzymatic Depletion Studies 23

3.6 Mouse Knockout Studies 23

4. Mechanical and Compositional Variations in Tendons with Different Functions 24

4.1 Variations in Tendon Mechanical Properties According to Tendon Function 24

4.2 Whole Tendon Properties 24

4.3 Variations in Fascicle-Level Mechanical Properties 25

4.4 Variations in Mechanical Properties at the Fiber (Microscale) Level 26

4.5 Variations in Mechanical Properties at the Fibril (Nanoscale) Level 27

4.6 Variations in Tendon Composition According to Tendon Function 28

4.7 Variation in Tendon Collagen 28

4.8 Variation in Collagen Cross-Links 29

4.9 Variation in Collagen Aggregates 30

4.10 Variation in Noncollagenous Components 31

4.11 Variation in Muscle–Tendon Relationship 32

4.12 Variation in Cell Density 33

4.13 Differences in Gene Expression 33

4.14 Differences in Matrix Turnover 34

4.15 Adaptability and Cell-Mediated Behavior 34

List of Abbreviations 35

Glossary 35

References 35

1. Tendon Structure and Composition

Tendons are fibrous soft tissue structures that connect muscle to bone. Their primary function is to act as passive, relatively inelastic structures, to allow force from muscle to be applied to bone. However, specific tendons, for example, the equine superficial digital flexor tendon (SDFT) and the human Achilles tendon, have additional functional specializations to allow energy storage [1]. They act like highly adapted elastic springs that stretch and store energy, which they can then return to the system through elastic recoil, to improve locomotory efficiency. This variability in tendon function requires differences in tendon structure.

The main component of tendon is water, which makes up 55–70% of the wet weight of a tendon. The major molecular components of the tendon extracellular matrix are collagens, which make up 60–85% of the tendon dry weight [2]. Tendons have a hierarchical organization, with the highly aligned collagen fibers arranged in a longitudinal manner, parallel to the mechanical axis, to develop a structure that has a high tensile strength (Figure 1). Each level of this collagen-rich hierarchy is interspersed with varying amounts of noncollagenous extracellular matrix [3,4].

Figure 1  Schematic showing the hierarchical structure of tendon, in which collagen molecules assemble to form subunits of increasing diameter.

While tendons may appear fairly similar on a large scale, there are consistent structural adaptations that allow tendons to achieve their specific functional specialization. Functional specialization is described later in the chapter; the general hierarchical arrangement of the matrix is first described.

When tendons are sectioned, the first subunit, large enough to be discernible by eye, is the fascicle, which consists of numerous collagen fibers bound together (Figure 2). Fascicles are irregular in shape and vary in diameter, which ranges from 150 to 500  μm. Surrounding the fascicles is a connective tissue compartment termed the interfascicular matrix (IFM). The IFM, sometimes referred to as endotenon, is highly important for tendons which are specialized for energy storage [3]. Each fascicle is composed of numerous collagen fibers that are packed together; collagen fibers are made up of collagen fibrils, with collagen fibrils made up of microfibrils. Collagen fibrils range in diameter from about 10  nm to approximately 500  nm [5]. Collagen fibrils are stabilized by specific cross-links that are intermolecular and bind adjacent microfibrils together [6]. Ultimately microfibrils are made up from groupings of most likely five collagen molecules linked by the same intermolecular cross-links as above and are often referred to as pentafibrils. Collagen molecules align longitudinally with a well-characterized quarter-staggered arrangement relative to an adjacent molecule. A gap of approximately 40  nm remains between the ends of each molecule. This arrangement results in a banded appearance of collagen fibrils or periodicity, which repeats every 67  nm and is known as the D period. The overlap region represents 0.4D and the gap region 0.6D.

The periphery of the tendon is encircled by a connective tissue sheath called the epitenon, which connects with the IFM. Away from joints, tendons are further surrounded by loose connective tissue, termed the paratenon, to facilitate tendon movement in their subcutaneous position. Where tendons pass over joints, they often are contained within a synovial sheath to facilitate and lubricate tendon movement.

Figure 2  A cross-sectional image of an equine superficial digital flexor tendon taken in the mid-metacarpal region. The fascicular arrangement of the tendon can be seen, creating an obvious honeycomb arrangement to the tendon, with each fascicle delineated by interfascicular matrix (IFM).

Arranging collagen in a highly aligned manner enables its direct loading in tension and ensures the tendon has sufficient tensile strength, while the inclusion of noncollagenous matrix provides a more viscous, time-dependent element to tendon behavior. However, regions of tendon require different properties, and tendon structure and composition often varies longitudinally within a tendon. Regions of tendons that experience predominant tensile loads contain highly aligned collagen and low levels of proteoglycans (PGs). However, regions of tendon that experience compressive loads, for example, where a tendon wraps around a joint, will contain a relative abundance of PGs and other proteins which are more cartilage-like. This higher abundance of PGs, such as aggrecan, increases tissue water content thereby providing increased stiffness and resistance to compression [7–9].

1.1. Collagens

The major collagen in tendon is Type I collagen, which makes up approximately 90% of the total collagen content. There are a number of other collagens that are present in tendon, in varying but small amounts, including collagen types III, V, VI, XI, XII, and XIV [10–13]. The fibrillar collagens (I, III, V, and XI) are dominated by long triple helical regions and are formed of three polypeptide chains; type I collagen consists of two α1 chains and one α2 chain and therefore there are two genes involved in collagen synthesis, one that codes for the α1 chain (Col1A1) and one for the α2 chain (Col1A2). Collagen-III comprises up to 10% of the collagen content of normal tendon and is thought to be essential for normal collagen fibrillogenesis and regulates the size of collagen type I fibrils [14]. Collagen-V is predominantly located in the core of collagen-I fibrils, and is thought to provide a template for fibrillogenesis [15]. Type VI collagen, which is a nonfibrillar collagen, has a characteristic pericellular distribution. Type XII and XIV collagens are fibril-associated collagens, which have interrupted triple helices and are closely associated with type I collagen. They provide a molecular bridge between fibrillar collagens and other matrix molecules. Type XII collagen has an important role in stabilizing collagen fibers during development, while type XIV collagen limits collagen fibril diameter during development [13].

1.2. Proteoglycans

PGs are a class of glycoproteins where a core protein is attached to one or more polysaccharide chains containing amino sugars. These chains are commonly referred to as glycosaminoglycan (GAG) side chains. In the tensile region of tendons, the majority of PGs present are small leucine-rich proteoglycans (SLRPs), which include molecules such as decorin, biglycan, fibromodulin, and lumican. In addition there are large aggregating PGs present within tendon; for example, aggrecan tends to be preferentially expressed in the compressive regions of the tendon, where a fibrochondrogenic phenotype predominates [16]. A similar large aggregating PG, versican, has been shown to be localized within the IFM with a predominantly pericellular distribution. Versican also interacts with elastic fibers, and is thought to contribute to the structural properties of the IFM [17,18]. However, the precise role of the large PGs in tendon function is unknown.

Figure 3  The relationship between collagen and decorin. The horseshoe-shaped decorin core protein (in white) is noncovalently bonded to a collagen molecule (in black), with a single covalently bound glycosaminoglycan side chain (jagged black line) sticking out from the structure. A 3-D spatial arrangement of the complex is shown. Reproduced from Vesentini et al. (2005) [100] with permission from Elsevier.

Decorin is the most abundant SLRP in tendon and accounts for approximately 80% of the total PG content. Decorin is a horseshoe-shaped protein, which binds noncovalently to a specific region of the collagen fibril. Attached to decorin is a single dermatan or chondroitin sulfate side chain, which binds to one edge of the core protein so that the GAG chain aligns parallel or perpendicular to the long axis of the collagen fibril. The side chain is then able to interact with the side chain of a decorin molecule bound to an adjacent collagen molecule, thus forming an interfibrillar bridge between adjacent fibrils (Figure 3). Decorin is found in both the IFM and between collagen fibrils within fascicles [3]. Biglycan similarly contains dermatan or chondroitin sulfate side chains and binds to the collagen fibril at a similar site to decorin. Lumican and fibromodulin have keratan sulfate side chains and share binding sites, distinct to the decorin-binding site, on the collagen fibril [3].

It has been postulated that SLRPs (and particularly decorin) may contribute to tendon mechanical properties, by transferring strain between adjacent discontinuous collagen fibrils. While each molecular bond between adjacent decorin side chains is relatively weak, when combined together they may be able to reach significant magnitude to transfer forces between fibrils. This is somewhat controversial, and some studies have questioned whether the interactions are sufficient to provide such force transfer. Further whether collagen fibrils are discontinuous is uncertain [3]. SLRPs are also considered to have a key role in tendon development, particularly relating to modulating the collagenous matrix during development. Most studies have focused on the role of decorin in tendon development, since it is the most abundant SLRP in tendon. Decorin has a role in aligning and stabilizing collagen fibrils during development, as well as inhibiting their lateral fusion, thereby regulating collagen fibril diameter. Other SLRPs also have a role in collagen fibrillogenesis; biglycan has a similar role in fibrillogenesis to decorin, and it has been proposed that both fibromodulin and lumican also have a similar role in inhibiting collagen fibril fusion [3].

Lubricin (also known as superficial zone PG or PRG4) is a large PG, which has many characteristics similar to mucins. Lubricin has been shown to be preferentially expressed on the tendon surface, in the IFM, and in the compressive regions of tendons. It has a role in lubricating tendons where surfaces need to glide over each other. It has a similar role in articular cartilage where it is preferentially expressed in the surface zone of cartilage [3,19].

1.3. Glycoproteins and Other Molecules

Glycoproteins consist of proteins covalently linked to a carbohydrate, which ranges from monosaccharide to polysaccharides. The most abundant glycoprotein in tendon is cartilage oligomeric matrix protein (COMP) which is a large pentameric protein consisting of five subunit arms arranged around a central cylinder [20]. Its function in tendon is uncertain; COMP-null knockout mice demonstrate no tendon abnormalities [21]. Mutations of the COMP gene are associated with pseudoachondroplasia, a disease characterized by joint laxity and other skeletal abnormalities, although the mechanisms leading to these pathologies are uncertain [22].

Tenascin-C is an extracellular matrix glycoprotein, which is present at low levels in mature musculoskeletal tissues, but is highly expressed in immature tissue as well as in pathology. Its function in tendon is unclear but it is present at the highest concentrations where tendons experience the highest mechanical forces, so it has been hypothesized that it has a role in providing tissue elasticity. Further, tenascin-C has been shown to be modulated by mechanical loading in rat Achilles tendon, with levels being increased by treadmill running, and decreased by limb immobilization [23], in a model for human Achilles tendon exercise.

Elastic fibers consist of a central core of elastin, surrounded by a sheath of polymers of fibrillin 1 and 2 and other associated proteins, which are known as microfibrils. The elastic fiber commonly associates with other molecules including decorin and biglycan. Elastin has been reported to be present in tendon at concentrations ranging from 1% to 10% of the tendon dry weight. While its role in tendon is currently undetermined, elastic fibers produce high elasticity, are highly fatigue resistant, and have the capacity for energy storage [3]. Localization of elastin in tendons is uncertain, but in canine cruciate ligaments, elastin has been found to be situated between fascicles [24]. Rat-tail tendon fascicles are similarly surrounded by a thin sheath of elastin [25]. Further in bovine flexor tendon, elastic fibers are broadly distributed throughout tendon but specifically localized around cells, as well as transversely between fascicles [26].

1.4. Cells

The extracellular matrix of the tendon is maintained and turned over by tenocytes (TCs), which are present in low density within the tendon (Figure 4). Cells in tendon either reside between the collagen fibers within the fascicles (intrafascicular TCs) or are grouped together in the IFM between the collagenous fascicles (interfascicular TCs). Intrafascicular TCs are elongated cells with extended nuclei and have a complex network of cytoplasmic processes, which extend through the matrix and connect with adjacent cells via gap junctions [27] while interfascicular TCs tend to have a plumper more rounded appearance. TCs are morphologically heterogeneous and the phenotypic distinction between TCs of different appearance is uncertain. In general tendon cell phenotype is poorly understood and defined. The interfascicular space also contains distinct vascular elements.

Figure 4  (A and B) Longitudinal H&E sections of equine superficial digital flexor tendon identifying the collagen-rich fascicles, interspersed with the highly cellular interfascicular matrix (IFM). IFM cells are heterogeneous in appearance, and contain obvious vascular structures.

1.5. Bone Insertion

At the point, where tendon inserts onto bone, a highly specialized interface develops, known as the enthesis. This structure is complex anatomically and biomechanically as it provides the interface between an elastic tendon (200 MPa tensile modulus) and the rigid and stiff bone (20 GPa tensile modulus) [28]. There are two forms of enthesis, fibrous and fibrocartilaginous. Fibrous entheses occur when the tendon is incorporated into the periosteum of the bone during development, with direct insertion of a tendon onto the metaphysis or epiphysis of a long bone [28]. A fibrocartilaginous enthesis occurs at the attachment of the tendon to the epiphysis or apophysis of bone. Such attachments have a complex hierarchical transitional organization and consist of four distinct zones [29]. Zone 1 consists of tendon proper, with longitudinally arranged tendon fibers analogous to normal tendon; zone 2 is made of unmineralized fibrocartilaginous tissue, which contains cartilage-specific collagens, such as collagen-II, and abundant PGs such as decorin and aggrecan; zone 3 consists of mineralized fibrocartilage, and contains varying amounts of bone mineral as well as collagens associated with endochondral ossification, such as collagen-X; zone 4 is consistent with bone. These zones show a gradual transition rather than abrupt boundaries [30]. Despite the complex arrangement of this insertional site to mitigate stress concentrations, pathology at this site is common [28].

1.6. Myotendinous Junction

The point at which tendon connects to muscle is a highly specialized region known as the myotendinous junction (MTJ). Unlike the bony insertion, the transition between muscle and tendon is not gradual, but occurs abruptly. However, the muscle cell membrane (sarcolemma) and tendon collagen fibers interdigitate via finger-like processes, increasing the interface area between muscle and bone, to distribute stress over a wider area and allow the MTJ to resist higher forces. Like the enthesis, the MTJ is prone to injury [31].

2. Tendon Mechanics

Collagen fibrils are one of nature’s main load-bearing structures, and their well-ordered arrangement within tendon results in a material with highly anisotropic mechanical properties. Such mechanics are important for function, ensuring tendon is stiff along its long axis and able to withstand its predominantly uniaxial load environment, transferring muscle forces along the tendon length to the skeleton.

Collagen is surrounded by PG-rich matrix at each level of the hierarchy, to make a multilevel fiber composite material. All connective tissues are composites, combining the properties of more than one component, and providing a comparatively simple method of altering tissue mechanics through manipulation of the composition and/or organization of the composite components. The aligned fiber composite structure of tendon ensures a flexible and damage-resistant tissue, where damage to a single area of the tendon does not easily propagate across the tissue.

In many ways, rope provides a good analogy for tendon; while very strong under tension applied along the long axis, it cannot sustain compression longitudinally and will simply buckle. Further, while both compressive and tensile strains can be sustained in the transverse direction, the tendon is an order of magnitude less stiff and strong in this direction, and testing in tension in this plane is difficult to achieve [7]. Indeed, if the tendon must withstand transverse compression within its physiological role, significant matrix adaptations are seen, as previously described.

There have been numerous studies investigating the mechanical properties of tendon over the last 50–60  years. Unsurprisingly, by far the majority of these have been carried out in tension along the tendon long axis, as this best represents physiological loading for tendon and is by far the simplest loading mode for the tissue. This chapter will subsequently focus in this area. Data can be grouped in many different ways, but one obvious distinction is between in vivo and in vitro analysis methods, for which a range of time-dependent and quasi-static testing protocols have been used to elucidate different tendon material properties.

2.1. In Vitro Mechanical Testing

Early studies were generally in vitro in nature, where it is reasonably simple to grip an isolated tendon sample and stretch it to failure to investigate its mechanical response. The simplest type of uniaxial tensile mechanical test is a quasi-static test, in which a sample is pulled to failure at a constant speed. The applied extension and resulting force are both recorded, and subsequently plotted as a force–extension curve describing the tendon mechanical response (Figure 5—inset). The slope of this curve describes how stiff the sample is, with a steeper gradient denoting greater forces needed to extend the sample, hence a stiffer tendon. However, tendon force–extension characteristics are rarely displayed directly, as both the amount of force a tendon can withstand and the amount it will stretch are dependent on its dimensions. Instead, stress–strain data are generally provided, in which force and extension data are both normalized for tendon dimensions to give generic material properties for a tissue (see Figure 5 for an explanation of stress and strain normalization methods). Such an approach is used across engineering, and from the resulting material stress–strain curve, the material stiffness (termed the modulus) can be found from the gradient of the curve. The idea of using such an approach is that the failure stress, failure strain, and modulus of any single material should be identical, irrespective of sample size you start with. While this works very well for homogeneous engineering materials such as steel, it is not such a simple relationship in composites, and significant variability remains in reported tendon mechanical properties.

The schematic stress–strain curve for a tendon shown in Figure 5 highlights the typical nonlinear mechanical response of tendon, in which four different regions can be identified; the toe region, heel region, linear region, and failure region [32]. The initial low stiffness behavior of tendon in the toe region results from the straightening of collagen crimp, and some reordering of the collagen so it is fully aligned in the loading direction [33]. This is followed by a heel region, in which stiffness begins to rise. This incorporates both molecular level reordering of the collagen molecules, straightening kinks in the gap region between the collagen molecules that constitute fibrils [34], and also a gradual direct loading of different collagen units once crimp had straightened. The application of further strain leads to the high stiffness linear region of the curve, in which the collagenous units of the tendon are directly loaded. There has been considerable debate as to how tendon extends in this region, but like any other fiber composite material, extension occurs through a combination of extension and sliding of the collagen units within their surrounding matrix at every level of the tendon hierarchical structure [35]. As further extension is applied to tendon, failure begins. This is usually seen as a steady pulling apart of the collagen fibers within the tendon, often resulting in a surprisingly gradual decrease in the force, as opposed to a sudden breakage [33].

Figure 5  Schematic stress–strain curve for tendon, highlighting the regions of the curve, and typical stress and strain values through each region for a range of tendons. The inset (top left) shows a force–extension curve, obtained from any in vitro mechanical test of tendon, with the main graph showing how stress, strain, and modulus are determined from these data.

The concept of calculating a single value of modulus within the linear region of the stress–strain curve is in reality difficult, as a careful look at a tendon stress–strain curve will highlight that the gradient of the curve is in fact continually changing. It is possible to draw a continual modulus curve, to see this behavior more clearly (Figure 6). Typically, a maximum modulus value is reported for a sample. Identifying the location of the maximum modulus also provides insight into the yield stress of a sample; as the modulus begins to fall, the sample has begun to fail.

Figure 6  (A and B) Schematic stress–strain curve for tendon (A), from which a continual modulus curve is drawn (B), calculating the gradient of the stress–strain curve over each 10   data points. The stress–strain is close to linear in the region delineated by dotted lines, in which the modulus curve is close to a straight line. The maximum modulus is easily identified from the peak value in the continual modulus curve.

The quasi-static mechanical properties of tendons vary considerably, so Figure 5 gives typically reported ranges for tendon behavior [36]. While some of the variation is a result of different testing protocols and testing artifacts between studies, there are clear differences in the properties of different tendons, related to differences in their required function. This will be reviewed later in the chapter, providing more details on the specific mechanical characteristics of functionally distinct tendons.

2.2. In Vivo Mechanical Testing

More recent mechanical characterization methods have focused on an in vivo analysis of tendon mechanics. While such an approach overcomes the limitations associated with gripping samples and maintaining appropriate hydration within an artificial environment, it introduces a substantial range of other potential errors and estimations. Indeed, as with all experimental procedures, it is important to realize that all mechanical testing methodologies bring their own potential errors, and data resulting from any experiment should be viewed with some caution and an understanding of the strengths and limitations of the approach.

Any mechanical test requires measures of the relationships between applied force and material extension, measures which can be approximated in vivo within a motion analysis laboratory, with the use of motion markers, a force plate, and ultrasound imaging. Of course tests-to-failure cannot be carried out in vivo, so typically in vivo tests aim to establish a tendon modulus.

The large majority of in vivo studies to date have investigated Achilles tendon mechanics, so the methodology for this application is briefly reviewed; however, the methodology can of course be adapted to investigate some other tendons, the most popular of which has been the patellar tendon. Typically, Achilles tendon mechanical properties are investigated as the subject loads the tendon by engaging the triceps surae muscle within the laboratory. As the muscles apply a force to the tendon, ultrasound is used to measure the resulting tendon stretch [37]. The most controlled method of carrying out this tendon loading is with an isometric contraction, where the complete tendon–muscle complex is constrained to remain still, so it does not change length during the muscle contraction. This can be achieved by tightly securing the leg in a dynamometer (Figure 7).

Figure 7  Measuring tendon mechanical behavior in vivo. (A) Schematic showing a leg strapped into a dynamometer. The subject uses the triceps surae to push against the dynamometer plate, recording force applied through the unit. Tendon extension is measured by tracking movement of the myotendinous junction (MTJ) and the calcaneus. (B) The MTJ is tracked with an ultrasound probe, allowing its displacement to be tracked during loading. Combining force and extension readings, a tendon loading curve can be acquired.

The force through the tendon is estimated from the force recorded on the dynamometer, treating the lower leg as a moment arm, and calculating the torque from the dynamometer force multiplied by the distance from point of contact (ball of foot) to the Achilles (Figure 7). In order to measure tendon extension, ultrasound imaging has traditionally been used [37]. This limits analysis to superficial tendons, and perhaps contributes to the particular focus of in vivo studies on the Achilles or patellar tendons. Ultrasound tracks the movement of the tendon-to-muscle interface within the calf (Figure 7), from which tendon length and extension can be established during movement. Motion analysis markers are usually placed on the calcaneus, to ensure its location is known and to check it does not move during measurements. Traditionally, a single value of force and extension has been collected around the point of maximum voluntary contraction of the muscle, and stiffness or modulus estimated by drawing a line from that point to zero (known as a secant modulus).

Adaptations and developments to these methodologies are continually proposed to try and improve the accuracy of in vivo measurements. For example, automated tracking of the MTJ throughout all the frames of ultrasound movies of tendon loading can now be carried out, enabling force–extension data to be continually collected and plotted, with curve fitting between data points able to reduce random tracking errors. This provides a considerably more representative measure of stiffness during the tendon loading range [38].

Recent studies have also begun to investigate functional movements, in which the Achilles is loaded as in exercise. Forces in this instance can be acquired from a force plate embedded in the laboratory floor, while motion markers can track tendon length change [38]. The difficulties of this approach lie in the need to carefully track the position of both the heel and the MTJ. The ultrasound probe must be strapped to the leg in order to record movement of the MTJ, while the positions of both the calcaneus (at the bony end of the tendon), and the location of the ultrasound probe must be established in 3D, if tendon length changes are to be established during movement.

2.3. Viscoelasticity

Tendon is a viscoelastic material, meaning it combines viscous and elastic behaviors, and quasi-static tests in isolation will not give a complete picture of tendon mechanical behavior. Indeed, the viscoelastic behavior of tendon can even make quasi-static mechanical testing complex, as the stiffness of viscoelastic materials is strain-rate-dependent, with faster loading speeds indicating a stiffer material.

A viscoelastic material is one in which the response to mechanical testing incorporates a time element. It is possible to compare a viscoelastic material such as tendon, with an elastic material such as a rubber band (Figure 8). If you stretch an elastic material and then remove the applied strain (Figure 8(A); column 1), it will follow an identical loading and unloading curve, meaning the relationship between force and extension in the material is consistent (Figure 8(A); column 2). By contrast, a viscoelastic material incorporates a time element to its response, so the unloading curve does not follow the loading curve, and the material will not return to its starting dimensions immediately upon removing the applied deformation (Figure 8(A); column 3). This behavior is termed hysteresis, and the area between the loading and unloading curve denotes the amount of energy lost during the cycle. In reality, no material is perfectly elastic, but in viscoelastic materials the amount of hysteresis is notable and there is clear energy loss with loading. Hysteresis is generally thought to occur as a result of water movement through the tissue and reordering of the multilevel fiber composite structure [39].

Figure 8  Schematic describing how elastic and viscoelastic materials respond to different applied load conditions, with the first column showing loading conditions and the second two columns the response of first an elastic, then a viscoelastic material. (A) A single load–unload cycle: An elastic material shows immediate recovery from loading and identical loading and unloading curves, while a viscoelastic material does not return to its original dimensions immediately. Viscoelastic materials lose energy during the cycle and take time for their dimensions to return to starting conditions. (B) Stress-relaxation: when an elastic material is loaded and held at a selected strain, the stress remains constant and conditions static until the strain is removed. In a viscoelastic material, water movement and molecular rearrangement contribute to a steady drop in the material stress over time. (C) Creep: when an elastic material is held under a selected stress, the length remains constant and conditions static until the load is removed. A viscoelastic material will gradually extend (creep) under the load until it eventually ruptures.

Another mechanism for investigating viscoelastic tissue behavior is to carry out time-dependent mechanical tests. These tests apply and hold a constant value of either extension or force to a sample, to see how it responds over time. The simplest test to perform is a stress relaxation test, in which a sample is strained by a certain amount and held, monitoring how the stress in the sample associated with that strain gradually reduces over time (Figure 8(B)). An elastic material will show no change in stress with time (Figure 8(B); column 2), while a viscoelastic material will steadily reorganize in attempt to reduce the stress until it reaches an equilibrium state (Figure 8(B); column 3). Measures of the rate of relaxation and equilibrium conditions are often quoted to characterize the degree of viscoelasticity.

Alternatively, it is possible to test a sample in creep, whereby a constant load is applied, and the steady elongation of the sample monitored (Figure 8(C)). While an elastic material once again shows no changes with time (Figure 8(C); column 2), a viscoelastic material will steadily elongate until it